Malaria is an infectious disease transmitted by inoculation of infectious sporozoites from the Plasmodium parasite via Anopheles mosquito bites. There are currently an estimated 500 million annual cases of malaria globally, and infection is lethal in 1-3 million people per year in endemic regions such as sub-Saharan Africa. Among the worst affected are children under the age of 5 years, in which the disease comprises several syndromes such as acute respiratory disease, severe anemia or cerebral malaria. The life cycle of Plasmodium falciparum, the parasite causing the most severe form of human malaria, is complex and multifaceted, with local differences within the host tissues and differing morphology corresponding to differential antigen expression. Following sporozoite inoculation, the parasite transforms and expands in an obligate, clinically silent and uni-directional phase in the liver (Prudenco et al., 2006). The release of packages of membrane-enclosed merozoites, termed merosomes, marks the onset of malaria (Sturm et al., 2006) though the symptoms and pathology of malaria, typically recognised by its cyclic pattern of fevers and chills, is caused exclusively during the rapid asexual multiplication phase of the Plasmodium parasite inside erythrocytes (Haldar et al., 2007). Thus, only preventative treatments targeting the pre-erythrocytic stages have the potential to prevent disease and offer effective protection. Immunisation of both humans and rodents with gamma-irradiated P. falciparum sporozoites (RAS) is the only experimental vaccine that confers essentially complete protection and is the gold standard of protective immunity against mosquito-borne malaria transmission (Nussenzweig et al., 1967; Hoffman et al., 2002). Irradiation disrupts the parasite genetic repertoire and parasites arrest in the liver, where they persist for up to 6 months post-infection (Silvie et al., 2002). However, these parasites are genetically undefined and, until today, have been restricted to experimental studies only. Nonetheless, this model provides proof-of-principle that sterile immunity in man is possible, based on the random genetic attenuation obtained by irradiation. Recent work has elicited sterile immunity in rodent models by genetically attenuated parasites (GAPs) (Mueller et al., 2005 a/b; van Dijk et al., 2005; Tarun et al., 2007; Aly et al., 2008; Silvie et al., 2008). These GAPs arrest at the early liver stage and confer sustained and stage-specific immunity (Mueller et al., 2007; Jobe et al., 2007).
The challenge for malaria vaccination based on genetically attenuated parasites depends on translating the results acquired in the rodent malaria models to human malaria. This has been successfully performed by disrupting p52 in P. falciparum, an ortholog of the rodent parasite gene p36p, which has been recently shown to induce sterilising immunity against sporozoite induced malaria (van Schaijk et al., 2008; van Dijk et al., 2005; VanBuskirk et al., 2009). The immune mechanisms conferring resistance to attenuated sporozoites are thought to rely on both humoral and cellular arms of the immune system. Apoptotic RAS-infected hepatocytes lead to presentation of Plasmodium antigens to dendritic cells, likely eliciting the protection-conferring immune response (Leiriao et al., 2005). Primarily, protection has been thought to rely on CD8+ T cells as the cytotoxic effector cells (White et al., 1996; Bongfen et al., 2007) and CD4+ T cells providing aid for antibody and optimal memory CD8+ cytotoxic T lymphocyte activity (Carvalho et al., 2002). Sterile immunity can be achieved in the absence of CD8+ T cells, mediated solely by CD4+ T cells and class-II effector mechanisms (Oliveira et al., 2008). Interestingly, a recently published model has been described where primaquine phosphate, a drug that exclusively targets Plasmodium liver stages, has been utilised in vivo to elicit vaccine-like protective immunity against subsequent sporozoite-induced rodent malaria in the absence of persistent metabolically active liver stages essential for such a CD8+ T cell response (Putrianti et al., 2009). Moreover, the use of wildtype parasites together with prophylactic chemotherapy (such as Chloroquine) results in protective immunity in P. falciparum (Roestenberg et al., 2009). Nonetheless, it has been suggested that long-persisting RAS forms present parasite antigen via MHC class I that induces IFN-gamma-based inducible nitric oxide synthase (iNOS) production by infected hepatocytes (Klotz et al., 1995). The circumsporozoite protein (CSP), the major surface protein of sporozoites, has historically been considered a major antigen and vaccine candidate associated with the liver stages of Plasmodium. Several CSP-derived T cell epitopes have been identified to date, some associated with very high levels of protection, as measured by the reduction in liver stage burden following challenge of immunised mice with normal sporozoites. RTS,S/AS02A is a pre-erythrocytic vaccine candidate based on P. falciparum CSP developed collaboratively between GlaxoSmithKline (GSK) and the Malaria Vaccine Initiative (MVI) Programme for Appropriate Technology in Health (PATH). However, its results are rather disappointing, with efficacies of between 40 and 60% (Alonso et al., 2004; Alonso et al., 2005; Bejon et al., 2008, Epstein et al., 2011). Such partial protection suggests that CSP may not be the sole protective antigen in attenuated models. Indeed, there is no CSP epitope described in C57B1/6 mice, though in Balb/c there is a well-known and characterised T-cell epitope. Interestingly, a recent study employing a P. berghei parasite line expressing heterologous P. falciparum CSP demonstrated that sterile immunity can be achieved in the absence of a CSP-specific immune response and concluded that hitherto uncharacterised antigens, and not CSP, may be targeted to induce sterilising immunity (Gruner et al., 2007; Mauduit et al., 2010).
It was an object of the present invention to provide means for an effective malaria vaccine. More specifically, it was an object of the present invention to identify novel liver stage antigens that are protective (i.e. critical for immunity), and thus can be used for vaccination.